Sorting out mutation rates.
Sorting out mutation rates.
复制标题
整理突变率。
DOI：
10.1073/pnas.96.14.7617
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发表时间：
1999
影响因子：
11.1
通讯作者：
Foster,PL
中科院分区：
文献类型：
--
作者：
Foster,PL
布卢明顿的一家乡村俱乐部里，萨尔瓦多·卢里亚在观看一台（无疑是非法的）老虎机时顿悟，已经过去了世纪。卢里亚-德尔布吕克波动测试（Luria-Delbrück fluctuation test）实验与赫尔希-蔡斯实验、帕贾莫实验、克里克等人的三联体密码实验以及梅塞尔森-斯塔尔实验一起，沿着存放在高雅科学博物馆的分子生物学区。卢里亚-德尔布吕克实验之所以受到科学家，尤其是遗传学家的喜爱，是因为它证明了一个假设，即突变是在细胞非选择性生长过程中随机出现的，而没有任何物理证据。卢里亚和德尔布吕克推断，如果一个突变体在培养物生长的早期碰巧出现，它将产生一个由相同后代组成的大型克隆。因为这种早期突变体是罕见的，所以在足够多的平行培养物中，突变细菌的最终数量将具有“异常高方差的分布”（2）。卢里亚和德尔布吕克通过证明方差确实远远大于均值，证明了他们的观点。但是，50多年来一直令人着迷的是Luria-Delbrück分布本身。对于我们这些研究自发突变的人来说，理解如何从波动测试中推导出突变率是一种入门仪式（参见参考文献3）。因此，当被告知Luria-Delbrück实验固有的波动并不是确定突变率的好方法，而是一个需要克服的问题时，这是一个令人震惊的消息。解决这个问题的理论基础如下（表1）。在指数增长的群体达到足够的规模（1/突变率/细胞）后，新突变体的组合加上先前存在的突变体的增长导致突变体分数的不断增加。突变率等于这种增加除以一定的时间（通常是细胞世代）（2）。因此，确定突变率的最简单方法是测量在不断增长的群体中突变体比例的变化。然而，当种群达到所需的规模时，突变已经发生并污染了培养物（波动效应），因此信噪比低得不可能。在1999年6月8日出版的《会议记录》中，巴赫尔等人（1999年）发表了一项研究。(4)已经通过用细胞分选仪消除预先存在的（荧光）突变体解决了这个问题。所得到的培养物几乎没有突变体，然后在随后的生长过程中，以预测的恒定速率积累新的突变体。(4)对一种有趣的突变现象感兴趣，即活化B淋巴细胞中IG基因座的超突变。在B细胞成熟过程中，重排的V（D）J区域的突变率比基因组的其余部分高105至106倍。这种超突变产生碱基置换突变，需要顺式和反式作用元件。巴赫尔等人将突变的非荧光形式的绿色荧光蛋白（GFP）置于胸苷激酶启动子下游，该启动子位于带有IG重链大内含子增强子的质粒上。当稳定转化到前B细胞系中时，突变的GFP基因以高速率回复，产生荧光细胞。在
It has been over half a century since Salvador Luria experienced an epiphany while watching a (no doubt illegal) slot machine in a country club in Bloomington, IN (1). The experiment that resulted, the Luria–Delbrück fluctuation test, resides in the molecular biology wing of the Museum of Elegant Science along with the Hershey–Chase, the PaJaMo, the Crick et al. triplet code, and the Meselson–Stahl experiments. What endears the Luria–Delbrück experiment to scientists, particularly geneticists, is that it proved a hypothesis—that mutations arise at random during nonselective growth of cells—without any physical evidence whatsoever. Luria and Delbrück deduced that if a mutant happened to arise early during the growth of a culture, it would produce a large clone of identical descendants. Because such early mutants would be rare, the final numbers of mutant bacteria among a sufficiently large number of parallel cultures would have ‘‘a distribution with an abnormally high variance’’(2). By showing that the variance was, indeed, far greater than the mean, Luria and Delbrück proved their case. But, what has continued to fascinate for over 50 years is the Luria–Delbrück distribution itself. For those of us who study spontaneous mutation, understanding how to derive mutation rates from fluctuation tests is something of an initiation rite (see ref. 3 for examples). So, it comes as a shock to be told that the fluctuations intrinsic to the Luria–Delbrück experiment constitute not a brilliant way to determine mutation rates but a problem to be overcome (4). The theory underlying the solution to the problem is as follows (Table 1). After an exponentially growing population reaches a sufficient size (1/mutation rate per cell), the combination of new mutants plus the growth of preexisting ones results in a constant increase in the mutant fraction. The mutation rate is equal to this increase divided by some measure of time (conventionally cell generations)(2). Therefore, the simplest of all ways to determine a mutation rate is to measure the change in the mutant fraction in a growing population. However, by the time the population reaches the required size, mutations have already occurred and polluted the culture (the fluctuation effect), so that the signal-to-noise ratio is impossibly low. In the June 8, 1999 issue of the Proceedings, Bachl et al.(4) have solved the problem by eliminating preexisting (fluorescent) mutants with a cell sorter. The resulting cultures were nearly mutant free and then, during subsequent growth, accumulated new mutants at the predicted constant rate.Bachl et al.(4) are interested in a fascinating mutational phenomenon, hypermutation of the Ig locus in activated B-lymphocytes. During B-cell maturation, the rearranged V (D) J region is subjected to a mutation rate some 105-to 106-fold higher than the rest of the genome. This hypermutation produces base-substitution mutations and requires both cis-and trans-acting elements. Bachl et al. placed a mutant nonfluorescent version of the green fluorescent protein (GFP) downstream of the thymidine kinase promoter on a plasmid bearing the large intron enhancer of the Ig heavy chain. When stably transformed into a pre-B cell line, the mutant GFP gene reverted at a high rate, yielding fluorescent cells. At the